Anti-diabetic pancreatic beta cells with down-regulated SLC30A8 gene expression and application thereof
A gene expression, β-cell technology, applied in pancreatic cells, genetically modified cells, cells modified by introducing foreign genetic material, etc., to achieve the effect of improving sensitivity, promoting maturation, and highly anti-apoptotic ability
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Embodiment 1
[0032] Construction of Human Embryonic Stem Cell Line with SLC30A8 Gene Knockout
[0033] 1 Plasmid construction
[0034] (1) Plasmid design and sgRNA screening
[0035] Use the tool website (http: / / crispr.mit.edu / ) to design a 23bp sgRNA for the upstream and downstream of the exon near the 5' end of the human SLC30A8 gene, and insert it into the carrying In the vector of Cas9 protein gene and puromycin resistance gene. According to the sgRNA target position and off-target rate score, the following sgRNAs were screened out:
[0036] sgRNA1 (SEQ ID NO.1): GACTGGCGTGCTAGTGTACCTGG;
[0037] sgRNA2 (SEQ ID NO. 2): CGATGATCATCACAGTCGCCTGG.
[0038]The plasmid sequence is as follows (SEQ ID NO.3):
[0039]
[0040] (2) Plasmid transformation and sequencing
[0041] Thaw the chemically competent cells for cloning on ice, add 10 μL of the ligation product to 100 μL of competent cells, flick the tube wall to mix well, let stand on ice for 30 minutes, heat shock in a water bath at ...
Embodiment 2
[0056] Effect of SLC30A8 Gene Knockout on the Promotion of Insulin Secretion in β Cells
[0057] The ability to secrete insulin in response to glucose stimulation is an important indicator of pancreatic β-cell function. The islet β cells induced to differentiate and mature in Example 1 were used as the SLC30A8 gene knockout group (SLC30A8), and compared with the wild control (WT) β cells without gene editing, the in vivo and in vitro functions were detected by the following methods, and the research Effect of SLC30A8 Gene Knockout on Insulin Secretion
[0058] 1 In vitro function test
[0059] In vitro studies mainly detect the insulin synthesis and secretion function of mature islet β cells. In terms of insulin synthesis, we monitored the conversion efficiency of proinsulin to insulin: using the difference in the recognition sites of proinsulin and insulin antibodies, the proinsulin-positive and insulin-positive cells were screened by flow cytometry, and the statistics The...
Embodiment 3
[0068] Example 3 Study of SLC30A8 Gene Knockout on Beta Cell Resistance to Type 2 Diabetes
[0069] The islet β cells of the SLC30A8 gene knockout group induced to differentiate and mature in Example 1 and the wild control group were used to study the mechanism of the SLC30A8 gene knockout islet β cells resisting the onset of type 2 diabetes by the following method.
[0070] 1 In vitro simulation of lipotoxic β-cell apoptosis induced by palmitic acid
[0071]The SLC30A8 gene knockout group and the wild control group were respectively formed into two groups of control groups (Control, Ct) and two groups plus 1mM palmitic acid solution group (PA). After culturing, the cell spheres were taken out for Annexin V living cell staining (this dye can identify early apoptotic cells), and confocal microscopy was performed to count the expression of Annexin V. Such as Figure 5 As shown, the results showed that under palmitic acid stimulation, the number of early apoptotic β cells in th...
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